Formulation and Evaluation of Facial Cleanser Containing Clitoria Ternatea Extract

The skin, the largest organ of the human body, accounting for nearly 15% of total adult body weight.1 The integumentary system is formed by the skin and its derivative structures. The skin is composed of three layers: the epidermis, the dermis and subcutaneous tissue.2Skin has two main layers. The uppermost one is the epidermis. It is a stratified squamous keratinized epithelium. The dermis is the second one a subjacent fibr0us- collagenous-elastic tissue that hosts vessels, nerves and sensory receptors. It supports the epidermis. The subcutaneous tissue hypodermis is the deepest layer, which in most instances consists largely of pads of adipose tissue. It is attached to the deep fascia or periosteum.3,4 A cosmetic is a word derived from the Greek word 'kosmesticos' meaning adornment. From ancient period, plants have been the basis for medical treatment and such traditional medicine is practiced even today. It is estimated that 80% of the people worldwide rely on herbal medicines derived from natural source. The word cosmetics is derived from Greek word "Kosmos" defined as articles intended to be rubbed, poured, sprinkled or sprayed or introduced into or otherwise applied to human body or any part for cleansing, beautifying, promoting attractiveness or altering appearances.5,6,7 Creams are defined as "a semisolid dosage form containing one or more drug substances dissolved or dispersed in a suitable base" They are formulated using hydrophilic or hydrophobic bases to provide preparations that are essentially miscible with the skin secretion. Semisolid emulsions of either 0/W or W/O type emulsions.8 Cleansing cream or lotion is required for removal of facial make-up, surface grime, oil, and water and oil soluble soil efficiently, mainly form the face and throat.9 In this formulation, medicinal herbs Clitoria ternatea commonly called as the butterfly pea of family Fabaceae.10 Its extracts possess a wide range of pharmacological activities including antibacterial, anti-diabetic, anti-diarrheal, anti-fungal, anthelmintic, anti-inflammatory, antimicrobial, antioxidant, antipyretic activities, hypolipidemia, immunomodulatory and wound healing.11,12 The Clitoria ternatea is one of the traditional medicinal plants used as an herbal medicine used to boost or enhance skin health when applied topically, butterfly pea flower can prevent early sign of skin aging, such as loss of firmness, fine lines, uneven skin tone, texture, skin hydration and protect against sun damage it helps to treat rashes, swelling, itching, dermatitis or allergies affecting the skin, soothing effects on skin, maintain skin elasticity. This in turn cleanses the body from within, deeply brightens dull skin and removes dark spots and uneven skin tone. Based on this herbal plant, formulation of cleanser for skin.13

OBJECTIVES

• To formulate cleanser from the extract of Clitoria ternatea.
• To characterize the developed the Clitoria ternatea cleanser, suitable for topical application.
• To evaluate the parameters of prepared cleanser.
• To carry out the consistency and spreadablility study from the prepared cleanser.
• To determine anti-microbial activity by well diffusion method.

METHODOLOGY

Ingredients used/ chemicals used

Table No 1: Composition of ingredients category.14,15

Experimental methods

Preparation of flower extract and preliminary phytochemical investigation Aqueous extract was prepared by stirring 5 g of the dried (powdered) plant material (flowers of Clitoria ternatea) with 100 ml of deionized water for 10 minutes. Extract was filtered by using filter paper (Whatman filter paper no. 41) from which the filtrate was used for formulation. This filtrate was initially subjected for preliminary phytochemical testing to confirm the presence of alkaloids, flavonoids, Tannis, saponins, phenols.16,17

Phytochemicals tests

Testing for alkaloids

Mayer's test: Few drops of Mayer’s reagent were added to 1 ml of extract. A yellowish or white precipitate was formed, indicating the presence of alkaloids.

Dragendroff's test: Few drops of dragendroff's reagent were added to 1ml of extract. A red precipitate was formed, it indicating the presence of alkaloids.

Wagner's test: Few drops of Wagner’s reagent were added to 1ml of extract. A reddish-brown precipitate was formed, it indicating the presence of alkaloids.

Test for phenol

Ferric Chloride test: Few drops of flower extract is added to 5 ml of distilled water and add 5% ferric chloride solution. Dark green, blue colour was formed, indicate the presence of phenolic compound.

Lead acetate test: Flower extract was added to 5 ml of distilled water and then add 10% lead acetate solution. Bulky white precipitate was formed, indicate the presence of phenolic compound.

Test for flavonoids

Alkaline reagent test: Flower extract was added to 10% ammonium hydroxide solution. A yellow fluorescence was formed. Indicate the presence of Flavonoids.

Shinoda test: Flower extract was dissolved in ethanol then add magnesium turnings and add the conc. HCL; Cherry red/pink colour was observed.

Test for Saponins

Foam test: Aqueous flower extract was shaken with distilled water (5 ml) for about 5 min. Persistence of frothing on warming confirmed the presence of saponins.

Test for Terpenoids

Salkowski test: Few drops of chloroform and conc. H2SO4 were added to 1ml of extract. A red precipitate was formed, it indicating the presence of terpenoids.

Test for steroids

Salkowski test: Few drops of chloroform and conc. H2SO4 were added to 1ml of extract. A reddish-brown precipitate was formed, it indicating the presence of steroids.18,19

Formulation and evaluation of cleanser

The oil phase (coco butter, bees wax, liquid paraffin, span derivatives) and aqueous phase (borax, methyl paraben, water flower extract, rose water, distilled water) were weighed separately and heated up to 60 degree Celsius in a water bath. The aqueous phase was added dropwise to oil phase with continuous stirring until a uniform cleanser was formed. Cleanser formulations were assessed for their physicochemical features like pH by using pre-calibrated pH meter and clarity by visual observation, viscosity test, stability test for 28 days are observed by visual.20,21

Table No   2: Composition of different formulation of cleanser.

Prepared gel was further evaluated for the following parameters:

Organoleptic Parameters: Formulations were evaluated based on their appearance, texture, color, odor and state. Texture was determined on the basis of grittiness/smoothness. Texture was found to be smooth; it can be spreadable and washable easily.

Measurement of pH: The pH of cleanser formulations was determined by using a digital pH meter. One gram of cleanser was dissolved in 100 ml of distilled water and stored for two hours. The measurement of pH of each formulation was done in triplicate, and average values were calculated.

Stability Study: The prepared cleanser formulation was filled in the container and stored at room temperature for 28 days. Formulations were observed for phase separation and color.

Homogeneity: The prepared cleanser formulation was examined by visual appearance of no spot and by touch.

Washability: Formulations were applied on the skin and then ease extend of washing with water was checked. Washability was checked by keeping applied skin area under the tap water for about 10 min.

Viscosity Study: The measurement of viscosity of the prepared cleanser was done with a Brook field Viscometer. The cleanser was rotated at 20 rpm using spindle no. 64. At each speed, the corresponding dial reading was noted.

Spreadability: Glass slides with standard dimension (length of 6.0 cm) were taken. Topical cleanser cream formulation was placed on the one side of the glass slide and sandwiched with the help of another slide. Remove the adhering cleanser on the outer surface of the glass slides by wiping. Slides are fixed in a stand that only upper slide to slip off freely without any disturbance by force of weight (20 g) tied to it. Time taken for the movement of upper slide to the distance of 6.0 cm was measured. Measurement of spreadability was done in triplicate and calculated by using the following formula:

Spreadability=(WeightxLength)/Time Where,

S=Spreadability

m=Weight tied to the upper slide (20 g)

L-Length of the glass (6.0 cm)

t=Time taken in seconds 59.22

Antimicrobial activity of Preparation of media

The required quantities of nutrient agar (2.8 g 100 ml) were prepared by dissolving it in distilled water in conical flasks. Media was sterilized in an autoclave at 15 psi pressure and 121°C for 15 min. After sterilization, nutrient agar media was poured aseptically into sterilized petri plates in a laminar flow hood. All the steps were performed in sterile environment in order to prevent contamination. The media was allowed to be solidified in petri plates for about an hour and then placed in an incubator at 37°C for 24 h. The next day, uncontaminated plates were used for culturing of the microorganisms after Incubation for 10 min. Antimicrobial activity was executed by agar well diffusion assay (antibiotic susceptibility testing) to compare the effect of cleanser and marketed cleanser formulation, extract of bpf against Pseudomonas aeruginosa, E Coli and Staphylococcus aureus. Sterilized MHA (Muller-Hinton agar) plates were prepared and incubated at 37ºC in bacterial incubator (Thermo Fisher Scientific, USA) overnight preceding to initiation of experiments to confirm contamination free plates. Before experimentation agar plates were dried 15 min and seeded with suspensions (100mcl) of test bacteria (Standardized by equating with 0.5 Mc Farland standard which equals to 15 X 108 CFU/ml) to make a uniform lawn of bacteria. 1ml of extract, 1ml of F5 and F6 formulations, blank, marketed formulation were sited in wells and incubated at 37ºC for 24hr. All experimentation were carried and outcomes were depicted in mean zone of inhibition(mm).23,24,25

RESULTS

1. Preparation of flower extract and preliminary phytochemical screening

Aqueous extract of the flower material by decoction method (dark purple color) was tested for its phytochemicals. Preliminary phytochemical screening of flower extracts discovered that the Clitoria ternatea consist of Alkaloids, flavonoids, tannins, saponins, phenols, terpenoids compounds. Results and observations of phytochemical tests are summarized in table.

Table no 3: Preliminary Phytochemical evaluation of flower extract

2. The absorption spectra of flower extract

Figure no 1: The absorption spectra of Clitoria ternatea flower extract

3. Fourier transform infrared spectroscopy (FT-IR)

Figure no 2: FT-IR spectrum of aqueous extract of Clitoria ternatea

Table no 3: FT-IR spectral data of aqueous extract of Clitoria ternatea and assignment of functional groups with respect to their wavenumbers.

Evaluation of Cleanser

1. Organoleptic evaluation:

Table no 4: Color, odor, texture, state of formulation F1 to F9.

2. Measurement of pH:

All the formulation within the range of 4.5 to 5.5

3.Washability test:

The washability of the formulations was evaluated by applying a small quantity of cleanser to the hand followed by rinsing with tap water. The results indicated that formulation F3 to F9 exhibited easy washability whereas formulation F1 and F2 demonstrated poor washability.

4. Spreadability test:

Table no 5: Spreadability of different formulations

5. Homogeneity

The prepared cleanser formulation was spread uniformly on a petri plate and examined by visually for presence of spots and by touch. Formulation F3 to F9 showed no spots and appeared uniform on plate but F1 and F2 showed some spots.

6. Phase separation

The phase separation by visually observing the breaking into two layers. Only F3 and F7, F8, F9 showed phase separation, while all other formulations remained stable without separation.  7.Viscosity test

Table no 6: determination of viscosity for different formulation

8. Antimicrobial activity

The antimicrobial activity study results of the formulated herbal cleanser showed antimicrobial activity against bacteria such as Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and E Coli. The antimicrobial study reveals that the herbal cleanser showed activity than the extract against all the pathogens. The antimicrobial activity of the study results are shown in (Figure).

Figure no 3: Antimicrobial activity of cleanser formulation F5 and F6 compare with marketed formulation, extract CT, blank against E Coli.

Table no 7: Zone of inhibition with E. coli bacterial strain.

Figure no 4: Antimicrobial activity of cleanser formulation F5 and F6 compare with marketed formulation, extract CT, blank against Pseudomonas aeruginosa.

Table no 8: Zone of inhibition with P. aeruginosa bacterial strain.

Figure no 5: Antimicrobial activity of cleanser formulation F5 and F6 compare with marketed formulation, extract CT, blank against Staphylococcus aureus.

Table no 9: Zone of inhibition with S. aureus bacterial strain.